Abstract:

The present invention relates to methods for preparing an artificial
immune system. The artificial immune system comprises a cell culture
comprising T cells, B cells and antigen-primed dendritic cells. The
artificial immune system of the present invention can be used for in
vitro testing of vaccines, adjuvants, immunotherapy candidates,
cosmetics, drugs, biologics and other chemicals.

Claims:

1. A method for immortalizing B cells monoclonal for an antigen,
comprising:(a) priming a population of dendritic cells with an
antigen;(b) adding the antigen-primed dendritic cells of (a) to a cell
culture comprising T cells and serum-free culture media;(c) priming a
population of B cells with the antigen;(d) adding the antigen-primed B
cells of (c) to the cell culture comprising T cells and antigen-primed
dendritic cells of (b);(e) culturing the cell culture of (d) under
conditions promoting production of antibodies by the antigen-primed B
cells;(f) identifying B cells in the culture of (e) that are monoclonal
for the antigen; and(g) isolating and immortalizing B cells identified in
(f) that are monoclonal for the antigen.

3. The method of claim 1, further comprising adding the antigen to the
cell culture (e).

4. A method for producing antibodies having binding specificity for an
antigen, comprising:(a) priming a population of dendritic cells with an
antigen;(b) adding the antigen-primed dendritic cells of (a) to a cell
culture comprising T cells and serum-free culture media;(c) priming a
population of B cells with the antigen;(d) adding the antigen-primed B
cells of (c) to the cell culture comprising T cells and antigen-primed
dendritic cells of (b); and(e) culturing the cell culture of (d) under
conditions promoting production of antibodies by the antigen-primed B
cells.

5. The method of claim 4, further comprising isolating antibodies specific
for the antigen from the culture of (e).

7. The method of claim 4, further comprising adding the antigen to the
cell culture (e).

8. A method for immortalizing B cells producing antibodies having binding
specificity for an antigen, comprising:(a) priming a population of
dendritic cells with an antigen;(b) adding the antigen-primed dendritic
cells of (a) to a cell culture comprising T cells and serum-free cell
culture media;(c) priming a population of B cells with the antigen;(d)
adding the antigen-primed B cells of (c) to the cell culture comprising T
cells and antigen-primed dendritic cells of (b);(e) culturing the cell
culture of (d) under conditions promoting production of antibodies by the
antigen-primed B cells;(f) identifying B cells producing antibodies
having binding specificity for the antigen in the culture of (e); and(g)
isolating and immortalizing B cells identified in (f) producing
antibodies having binding specificity for the antigen.

10. The method of claim 8, further comprising adding the antigen to the
cell culture of (e).

11. A method for producing a therapeutic composition comprising monoclonal
antibodies having binding specificity for an antigen, the method
comprising:(a) priming a population of dendritic cells with an
antigen;(b) adding the antigen-primed dendritic cells of (a) to a cell
culture comprising T cells and serum-free cell culture media;(c) priming
a population of B cells with the antigen;(d) adding the antigen-primed B
cells of (c) to the cell culture comprising T cells and antigen-primed
dendritic cells of (b);(e) culturing the cell culture of (d) under
conditions promoting production of antibodies by the antigen-primed B
cells;(f) identifying B cells in the culture of (e) that are monoclonal
for the antigen;(g) isolating and immortalizing B cells identified in (f)
that are monoclonal for the antigen; and(h) preparing a therapeutic
composition comprising monoclonal antibodies produced by the immortalized
B cells of (g) and a pharmaceutically acceptable carrier.

12. A method for producing a therapeutic composition comprising a
monoclonal antibody having binding specificity for an antigen, the method
comprising:(a) priming a population of dendritic cells with an
antigen;(b) adding the antigen-primed dendritic cells of (a) to a cell
culture comprising T cells and serum-free cell culture media;(c) priming
a population of B cells with the antigen;(d) adding the antigen-primed B
cells of (c) to the cell culture comprising T cells and antigen-primed
dendritic cells of (b);(e) culturing the cell culture of (d) under
conditions promoting production of antibodies by the antigen-primed B
cells;(f) identifying B cells in the culture of (e) that are monoclonal
for the antigen;(g) isolating and immortalizing B cells identified in (f)
that are monoclonal for the antigen; and(h) preparing a therapeutic
composition comprising monoclonal antibodies produced by one of the
immortalized B cells of (g) and a pharmaceutically acceptable carrier.

13. A method for producing a therapeutic composition comprising antibodies
having binding specificity for an antigen, the method comprising:(a)
priming a population of dendritic cells with an antigen;(b) adding the
antigen-primed dendritic cells of (a) to a cell culture comprising T
cells and serum-free cell culture media;(c) priming a population of B
cells with the antigen;(d) adding the antigen-primed B cells of (c) to
the cell culture comprising T cells and antigen-primed dendritic cells of
(b);(e) culturing the cell culture of (d) under conditions promoting
production of antibodies by the antigen-primed B cells;(f) identifying B
cells in the culture of (e) producing antibodies having binding
specificity for the antigen;(g) isolating and immortalizing B cells
identified in (f) having binding specificity for the antigen; and(h)
preparing a therapeutic composition comprising antibodies having binding
specificity for the antigen produced by the immortalized B cells of (g)
and a pharmaceutically acceptable carrier.

14. A method for preparing a therapeutic composition comprising antibodies
having binding specificity for an antigen, the method comprising:(a)
priming a population of dendritic cells with an antigen;(b) adding the
antigen-primed dendritic cells of (a) to a cell culture comprising T
cells and serum-free cell culture media;(c) priming a population of B
cells with the antigen;(d) adding the antigen-primed B cells of (c) to
the cell culture comprising T cells and antigen-primed dendritic cells of
(b);(e) culturing the cell culture of (d) under conditions promoting
production of antibodies by the antigen-primed B cells;(f) isolating
antibodies specific for the antigen from the culture of (e); and(g)
preparing a therapeutic composition comprising the isolated antibodies of
(f) and a pharmaceutically acceptable carrier.

Description:

CROSS REFERENCE TO RELATED CASES

[0001]This application is a divisional of U.S. application Ser. No.
11/594,172, filed Nov. 8, 2006, which is continuation-in-part of U.S.
application Ser. No. 11/453,046, filed Jun. 15, 2006, which is a
continuation-in-part of U.S. application Ser. No. 11/116,234, filed Apr.
28, 2005, which claims the benefit of U.S. Provisional Application Ser.
Nos. 60/565,846, filed Apr. 28, 2004, and 60/643,175, filed Jan. 13,
2005. This application also claims the benefit of priority of
International Application No. PCT/US05/014444, filed Apr. 28, 2005. Each
of these applications is hereby incorporated by reference in their
entirety

BACKGROUND OF THE INVENTION

[0002]1. Field of the Invention

[0003]The present invention is directed to a method for constructing an
integrated artificial human tissue construct system and, in particular,
construction of an integrated human immune system for in vitro testing of
vaccines, adjuvants, immunotherapy candidates, cosmetics, drugs,
biologics, and other chemicals. The artificial immune system of the
present invention is useful for assessing the interaction of substances
with the immune system, and thus can be used to accelerate and improve
the accuracy and predictability of, for example, vaccine, drug, biologic,
immunotherapy, cosmetic, and chemical development.

[0004]2. Background of the Technology

[0005]Despite the advent and promise of recent technologies, including
combinatorial chemistry, high-throughput screening, genomics, and
proteomics, the number of new drugs and vaccines reaching the market has
not increased. In fact, the attrition rate within drug discovery programs
exceeds 90%.

[0006]The introduction of these new (and expensive) technologies has not
reduced the lost opportunity costs associated with immunotherapy
development; rather, these costs have increased. Indeed, it is now
estimated that almost $1 billion is required to bring a new drug to the
market.

[0007]The development and biological testing of human vaccines has
traditionally relied on small animal models (e.g., mouse and rabbit
models) and then non-human primate models. However, such small animal
models are expensive and non-human primate models are both expensive and
precious. Furthermore, there are many issues regarding the value of such
animal studies in predicting outcomes in human studies.

[0008]A major problem remains the translation from test systems to human
immunology. Successful transfer between traditional testing systems and
human biology requires an intricate understanding of disease pathogenesis
and immunological responses at all levels. Given worldwide health
problems caused by known and emerging infectious agents and even
potential biological warfare pathogens, it is time for a fresh approach
to understanding disease pathogenesis, the development and rapid testing
of vaccines, and insights gathered from such work.

[0009]The body's distributed immune system can be roughly divided into
four distinct compartments: tissues and blood, mucosal tissues, body
cavities, and skin. Because of ease of study, most is known about the
tissue and blood compartment and its lymphoid tissues, the spleen and
lymph nodes.

[0010]The mammalian immune system uses two general adaptive mechanisms to
protect the body against environmental pathogens. When a pathogen-derived
molecule is encountered, the immune response becomes activated to ensure
protection against that pathogenic organism.

[0011]The first immune system mechanism is the non-specific (or innate)
inflammatory response. The innate immune system appears to recognize
specific molecules that are present on pathogens but not within the body
itself.

[0012]The second immune system mechanism is the specific or acquired (or
adaptive) immune response. Innate responses are fundamentally the same
for each injury or infection; in contrast, acquired responses are
custom-tailored to the pathogen in question. The acquired immune system
evolves a specific immunoglobulin (antibody) response to many different
molecules, or antigens, derived from the pathogen. In addition, a large
repertoire of T cell receptors (TCR) is sampled for their ability to bind
processed peptides from the antigens that are bound by major
histocompatibility complex (MHC) class I and II proteins on the surface
of antigen-presenting cells (APCs), such as dendritic cells (DCs).

[0013]Acquired immunity is mediated by specialized immune cells called B
and T lymphocytes (or simply B and T cells). Acquired immunity has
specific memory for specific antigens; repeated exposure to the same
antigen increases the memory response, which increases the level of
induced protection against that particular pathogen.

[0014]B cells produce and mediate their functions through the actions of
antibodies. B cell-dependent immune responses are referred to as "humoral
immunity" because antibodies are found in body fluids.

[0015]T cell-dependent immune responses are referred to as "cell-mediated
immunity," because effector activities are mediated directly by the local
actions of effector T cells. The local actions of effector T cells are
amplified through synergistic interactions between T cells and secondary
effector cells, such as activated macrophages. The result is that the
pathogen is killed and prevented from causing diseases.

[0016]The functional element of a mammalian lymph node is the follicle,
which develops a germinal center (GC) when stimulated by an antigen. The
GC is an active area within a lymph node, where important interactions
occur in the development of an effective humoral immune response. Upon
antigen stimulation, follicles are replicated and an active human lymph
node may have dozens of active follicles, with functioning GCs.
Interactions between B cells, T cells, and FDCs take place in GCs.

[0017]Various studies of GCs in vivo indicate that the many important
events occur there, including immunoglobulin (Ig) class switching, rapid
B cell proliferation (GC dark zone), production of B memory cells,
accumulation of select populations of antigen-specific T cells and B
cells, hypermutation, selection of somatically mutated B cells with high
affinity receptors, apoptosis of low affinity B cells, affinity
maturation, induction of secondary antibody responses, and regulation of
serum immunoglobulin G (IgG) with high affinity antibodies. Similarly,
data from in vitro GC models indicate that FDCs are involved in
stimulating B cell proliferation with mitogens and it can also be
demonstrated with antigen (Ag), promoting production of antibodies
including recall antibody responses, producing chemokines that attract B
cells and certain populations of T cells, and blocking apoptosis of B
cells.

[0018]Similar to pathogens, vaccines function by initiating an innate
immune response at the vaccination site and activating antigen-specific T
and B cells that can give rise to long term memory cells in secondary
lymphoid tissues. The precise interactions of the vaccine with cells at
the vaccination site and with T and B cells of the lymphoid tissues are
important to the ultimate success of the vaccine.

[0019]Almost all vaccines to infectious organisms were and continue to be
developed through the classical approach of generating an attenuated or
inactivated pathogen as the vaccine itself. This approach, however, fails
to take advantage of the recent explosion in our mechanistic
understanding of immunity. Rather, it remains an empirical approach that
consists of making variants of the pathogen and testing them for efficacy
in non-human animal models.

[0020]Advances in the design, creation and testing of more sophisticated
vaccines have been stalled for several reasons. First, only a small
number of vaccines can be tested in humans, because, understandably,
there is little societal tolerance for harmful side effects in healthy
people, especially children, exposed to experimental vaccines. With the
exception of cancer vaccine trials, this greatly limits the innovation
that can be allowed in the real world of human clinical trials. Second,
it remains challenging to predict which immunodominant epitopes are
optimal for induction of effective CD4.sup.+ and CD8.sup.+ T cell
responses and neutralizing B cell responses. Third, small animal testing,
followed by primate trials, has been the mainstay of vaccine development;
such approaches are limited by intrinsic differences between human and
non-human species, and ethical and cost considerations that restrict the
use of non-human primates. Consequently, there has been a slow
translation of basic knowledge to the clinic, but equally important, a
slow advance in the understanding of human immunity in vivo.

[0021]The artificial immune system (AIS) of the present invention can be
used to address this inability to test many novel vaccines in human
trials by instead using human tissues and cells in vitro. The AIS enables
rapid vaccine assessment in an in vitro model of human immunity. The AIS
provides an additional model for testing vaccines in addition to the
currently used animal models.

[0023]Nevertheless, none of these publications describe or suggest an
artificial (ex vivo) human cell-based, immune-responsive system
comprising a vaccination site (VS) and a lymphoid tissue equivalent
(LTE). The present invention comprises such a system and its use in
assessing the interaction of substances with the immune system.

[0030]FIG. 5: Shows an in vitro system representative of the physiological
state promotes stronger B cell proliferative and tetanus toxoid-specific
antibody responses, using a 2D co-culture of T and B cells and TT-pulsed
DCs.

[0031]FIG. 6: Depicts tetanus-specific antibody responses to a DTaP
(diphtheria and tetanus and acellular pertussis vaccine, adsorbed)
vaccine and a simple tetanus toxoid Antigen, using a 2D co-culture of T
and B cells and TT-pulsed DCs.

[0032]FIG. 7: Shows the influence of vaccine versus antigen in a lymphoid
tissue equivalent (LTE) for the same cell donor shown in FIG. 6.

[0038]FIG. 13: Shows in vitro the importance of the temporal sequence of
immunological events.

[0039]FIG. 14: Shows a rapid assay system for the development of primary B
cell responses.

DETAILED DESCRIPTION OF THE INVENTION

[0040]The present invention concerns the development of accurate,
predictive in vitro models to accelerate vaccine testing, allow
collection of more informative data that will aid in redesigning and
optimizing vaccine formulations before animal or clinical trials, and
raise the probability that a vaccine candidate will be successful in
human trials. More specifically, the present invention comprises
controlling the nature and state of the cells in the lymphoid tissue
equivalent (LTE, artificial lymph node) of the artificial immune system
(AIS).

[0041]The AIS can be used to test vaccines and other pharmaceuticals for
immune reactivity in a manner that is more predictive than animal
experiments. Consequently, it can provide valuable pre-clinical data
earlier in the research and development process. Antigenic molecules
introduced to the AIS are acquired by dendritic cells (DCs) at the
vaccination site (VS). The DCs are then transferred to the lymphoid
tissue equivalent (LTE), where they present the antigen to T cells,
activating their immune function. Activated helper T cells co-stimulate B
cells to induce antibody production, while activated cytotoxic T cells
lyse antigen-bearing cells. Solubilized antigen(s) can also be introduced
into the LTE to directly activate B cells for subsequent antibody
production.

[0042]While a number of published reports have demonstrated
antigen-specific B cell responses (to C. albicans, TT, and other
antigens) in vitro, these results are typically achieved by stimulating
and restimulating cultures of whole PBMCs with antigen and exogenous
factors to boost B cell proliferation and/or activation.

[0043]The present invention comprises the detection of antibody responses
using defined cultures of B cells, T cells, and DCs and optionally
follicular dendritic cells (FDCs), in 2-dimensional construct assay. The
presence of secondary cells provides a more physiological environment for
B cell activation and differentiation, such that artificial factors in
the cultures are not necessary to detect specific antibody responses. In
an embodiment of the present invention, the LTE comprises allogeneic T
cells. In another embodiment, the LTE comprises autologous T cells.

[0044]Using embodiments of the present invention, we have generated
antigen-specific B cell responses using a 2-dimensional (2D) co-culture
system comprising T cells, B cells, and antigen-pulsed DCs. In the
examples, responses were generated against tetanus toxoid (TT) and a
whole protein extract of Candida albicans (C. albicans). The results from
these examples show that culturing human T and B cells together in vitro
at a ˜1:1 ratio, versus the ratio of T and B cells naturally found
in the blood, gave stronger antigen responses, by both analysis of
activation and proliferation (flow cytometry) and antibody production
(ELISPOT). Although the preferred ratio of T cells:B cells is ˜1:1,
the ratio of T cells:B cells can range from ˜1:10 to ˜10:1.
In the cultures of the examples, "T cells" included both CD4.sup.+ and
CD8.sup.+ T cells. In peripheral blood, the T (total T cells):B cell
ratio is ˜7:1. In the lymph node, the T (total T cells):B cell
ratio is ˜1:1.6. In the germinal center, the T cell:B cell ratio is
˜1:8, and there, the T cells are primarily CD4.sup.+ T cells.

[0045]In the results of the experiments shown, engineered serum-free media
(X-VIVO) was used, though we have also used serum (e.g., human, bovine)
in other experiments (data not shown). Dendritic cells (DCs) were
generated from CD14-purified monocytes that were cultured for ˜7
days in X-VIVO 15 media, supplemented with GM-CSF (˜100 ng/ml) and
IL-4 (˜25 ng/ml). The cytokine-derived DCs were pulsed with antigen
or vaccine and then cocultured with T cells. After adding the
antigen-prepulsed dendritic cells to the T cell culture, B cells primed
with the same antigen used to prime the DCs are added to the cell
culture. After adding the antigen-primed B cells to the cell culture,
further soluble antigen can also be added. For PBMC cultures, either the
antigen was added to the assay, or antigen-pulsed DCs were added to the
assay. In FIGS. 1 to 9, antigen-pulsed DCs were added to the co-culture
of T and B cells, while soluble antigen was added to the PBMC cultures.
FIG. 9 shows a comparison of the co-culture to PBMCs, with antigen-pulsed
DCs added to both systems.

[0046]Antibodies specific for the antigen of interest can be isolated from
the resulting cell cultures of the present invention. Such antibodies can
be used for a variety of purposes, including in therapeutic and
diagnostic methods.

[0047]In addition, antigen-specific B cells can be isolated, cloned and
immortalized from the cell cultures of the present invention, and can
also be used in therapeutic and diagnostic methods.

[0048]Alternatively, all of the antibody-producing B cells are collected
en masse (without isolating/cloning individual B cells) for immortalizing
and producing a therapeutic.

[0049]Thus, the present invention also encompasses methods of producing a
therapeutic comprising antibodies or antibody-producing B cells in
combination with a pharmaceutically acceptable carrier.

EXAMPLES

[0050]These experiments provide a direct comparison of PBMCs versus a
co-culture of negatively selected T and B cells that were plated at a
˜1:1 ratio in--in these examples--a 96-well, round bottom plate.
All assays were harvested on day 7 of in vitro culture. All experiments
were analyzed by ELISPOT for antibody production and by flow cytometry
for proliferation, as determined by loss of CFSE. In the ELISPOT assays
because there were different ratios of T and B cells in the PBMC culture
compared with the TB-2D cultures, there were fewer B cells plated into
the ELISPOT wells. However, in the experiment in FIG. 4, the numbers of B
cells used in the ELISPOT experiments for both the PBMC and co-culture
assays were approximately equal. We determined the approximate number of
B cells in the ELISPOT wells by flow cytometry to enable comparisons.

[0051]These results show that culturing human T and B cells together in
vitro at a ˜1:1 ratio compared to the ratio of T and B cells
naturally found in the blood give stronger antigen responses, by both
analysis of activation and proliferation (flow cytometry) and antibody
production (ELISPOT). The results also show that co-culturing T cells and
antigen-primed dendritic cells, and subsequently adding antigen-primed B
cells, also gives stronger antigen responses, as indicated by antibody
production (ELISPOT).

[0053]PBMC versus co-culture, using a tetanus toxoid antigen. Even though
similar B cell proliferation responses were seen in PBMC and 2D T and B
cell co-cultures (FIGS. 2, 3), an improved tetanus toxoid-specific
antibody response was observed in a T and B cell co-culture LTE, as
compared with PBMC cultures (FIG. 4).

Example 2b

[0054]PBMC versus co-culture, using Candida albicans antigens. FIG. 9
shows C. albicans-specific ELISPOT data, comparing TB-2D to PBMCs. In
this experiment, DCs were pulsed with TT antigen only, but the ELISPOT
was conducted on both TT- and C. albicans-coated plates.

Example 2c

[0055]PBMC versus co-culture (FIG. 10). In this example we addressed the
question of what happens if we take cells from an apparent
"non-responder" and use only the GC cells from the leukocytes. Note the
response when some of the leukocytes are removed (FIG. 10);
non-responders in vitro now show an antibody response.

[0056]Here, we used human CD4.sup.+ T and B cells with FDCs and formed GCs
in vitro and then examined whether IgG production could be obtained
against a recall antigen. Specifically, we used tetanus toxoid (TT) in
these experiments and isolated human B cells and CD4.sup.+ T cells from
peripheral blood.

[0057]We observed IgG recall responses using only the T cells, B cells,
and FDCs that are typically found in GCs. In contrast, in the presence of
PBL cells not normally in found in GCs, no antibody response was
detectible in cells from some donors. These results show that removing
(not including) other cells, such NK cells, monocytes, and CD8.sup.+ T
cells, improved the IgG response.

[0059]Use of a vaccine to elicit in vitro immune responses in a co-culture
of T and B cells. DCs were pulsed with the vaccine or the tetanus toxoid
antigen and were then added to the co-culture of T and B cells.
Tripedia® (diphtheria and tetanus toxoids and acellular pertussis
vaccine, adsorbed; DTaP), for intramuscular use, is a sterile preparation
of diphtheria and tetanus toxoids adsorbed, with acellular pertussis
vaccine in an isotonic sodium chloride solution containing thimerosal
(preservative) and sodium phosphate (to control pH). After shaking, the
vaccine is a homogeneous white suspension. Tripedia® vaccine is
distributed by Aventis Pasteur Inc.

Example 5

[0060]To detect antigen-specific antibody responses, we developed an
ELISPOT approach to quantify B cell responses (antigen specificity) on a
per cell basis. In this example, T cells were cultured with B cells at a
˜1:1 ratio, with cytokine-derived DCs included at a DC:T and B
(total) cell ratio of ˜1:60. Soluble TT (˜1 μg/ml) or C.
albicans (˜10 μg/ml) was included for the entire 7-day culture,
while other wells received pokeweed mitogen (PWM; a strong, non-specific
lymphocyte stimulator) for the final 3 days of the culture.

[0061]On the seventh day, the lymphocytes were examined for marker
expression and CFSE profiles by flow cytometry and the frequency of TT
and C. albican-specific B cells was calculated by ELISPOT. Briefly,
˜30×103 total lymphocytes were plated in duplicate wells
of an ELISPOT plate that had been pre-coated with TT, C. albicans, or
anti-immunoglobulin (Ig, to gauge total antibody production).

[0062]The cells were then serially diluted five times at a ˜1:3
ratio and PWM was added to all wells to trigger antibody production. The
cells were then incubated for ˜5 hr at 37° C. in a 5%
CO2 incubator and washed away. Plate-bound antibody was detected
using techniques similar to those required for ELISA.

[0064]The lack of a robust response against TT was consistent with the
weak serum TT titer for this donor (˜4 μg/ml). As expected, PWM
triggered potent T and B cell proliferative responses, though not as many
divisions were seen as with specific antigen stimulation, likely because
the cells were only cultured with the mitogen for 3 days.

[0065]The specificity of the C. albicans-stimulated B cells was
demonstrated by ELIPSOT (FIG. 2). This experiment suggests that a
1×stimulation with C. albicans did give rise to a small population
of antibody-producing cells (˜0.2% of total B cells) that was not
detected in untreated cultures or those stimulated with TT (left and
middle wells). This discrepancy between the frequency of proliferating
cells and C. albicans-specific B cells detected by ELISPOT could be the
result of several factors. A likely explanation is that we used a crude
C. albicans whole antigen extract containing ˜19% carbohydrates (by
weight). While C. albicans polysaccharides are strong inducers of B cell
responses, only protein antigen-specific responses would be detected in
the ELISPOT assay.

Example 6

[0066]Tetanus-specific antibodies were detected in another ELISPOT
experiment where the cell donor's serum anti-tetanus level was higher (63
μg/ml), and DCs were cultivated in XVIVO-15 medium. All other
components, concentrations and ratios were left unchanged, except that of
the number of cells deposited per ELISPOT well was increased; the higher
number used was ˜1×105 cells/well.

[0067]In this experiment, both TT- and C. albicans-specific antibodies
were observed (up to 48 and 33 spots per well, respectively), although a
high level of non-specific response, especially in the presence of
CCL21/anti-CD40 additives, did not allow a firm conclusion in favor of
antigen-specific versus mitogenic activity.

Example 7

[0068]The specificity of the C. albicans-stimulated B cells was
demonstrated by ELIPSOT (FIG. 9) for both PBMC and 2D co-culture of T and
B cells with C. albicans-pulsed DCs added to both systems. This
experiment indicates that even if the PBMC cultures have antigen-pulsed
DCs added that the co-culture system shows a stronger antibody response,
as determined by ELISPOT.

Example 8

[0069]In vitro antigen-specific antibody response to influenza (FIG. 11)
and T and B cell proliferation induced by H1N1 influenza (FIG. 12). DCs
were treated (or not) with H1N1 (New Caledonia) influenza. 2D cultures of
DCs and T and B cells were stimulated (or not) with `soluble` H1N1
influenza. As can be seen, there was antigen-specific proliferation of T
and B lymphocytes and generation of antigen-specific antibody secreting B
lymphocytes (ELISPOT data). Note the largest (apparently synergistic)
response was observed when we pulsed the DCs with antigen and then added
soluble antigen to the DC/T and B cell cultures, to activate the B cells,
which are antigen-presenting cells (APCs). Again, the T and B cell
co-culture is superior to PBMC cultures.

Example 9

[0070]In this example, the antigens examined were tetanus toxoid (TT) and
a whole protein extract of Candida albicans.

[0072]In the first experiment, B cells were added at the same time (day 0)
in equal numbers to the T cells, DCs and antigen.

[0073]In the second experiment, antigen-primed B cells were added to the
T/DC cultures 3 days after the T/DC cultures were established.

[0074]As a control, cultures were also established in the absence of any
antigen.

[0075]Seven days following the addition of the antigen-primed B cells to
the T/DC cultures, the cells were harvested and analyzed for
antigen-specific B cells by ELISPOT assay.

[0076]As FIG. 13 illustrates, the immune (antibody) response was much
stronger in the case where the antigen-primed B cells were added to the
T/DC culture after 3 days. Similar results were obtained using cells
obtained form three independent blood donors.

Example 10

[0077]A rapid assay system for the development of primary B cell
responses. Antigen-primed dendritic cells (DCs) were cultured with
syngeneic B cells (˜2 to ˜2.5×106) in 24-well
plates and allogeneic CD4.sup.+ T cells at a 1:100 ratio (i.e., ˜2
to ˜2.5×104 allogeneic T cells). Additional antigen was
added (the antigens used were keyhole limpet hemocyanin (KLH) and the
anthrax recombinant protective antigen (rPa)). Fourteen days later, the
primary B cell response was assessed by ELISPOT and FACS. As FIG. 14
illustrates, the ELISPOT results show that, despite some background,
enhanced primary B cell responses (IgM) were observed to the antigens.

[0078]While the foregoing specification teaches the principles of the
present invention, with examples provided for the purpose of
illustration, it will be appreciated by one skilled in the art from
reading this disclosure that various changes in form and detail can be
made without departing from the true scope of the invention.